The transcriptional complex, hypoxia inducible factor (HIF), is a key regulator of oxygen homeostasis. Hypoxia induces the expression of genes participating in many cellular and physiological processes, including oxygen transport and iron metabolism, erythropoiesis, angiogenesis, glycolysis and glucose uptake, transcription, metabolism, pH regulation, growth-factor signaling, response to stress and cell adhesion. These gene products participate in either increasing oxygen delivery to hypoxic tissues or activating an alternative metabolic pathway (glycolysis) which does not require oxygen. Hypoxia-induced pathways, in addition to being required for normal cellular processes, can also aid tumor growth by allowing or aiding angiogenesis, immortalization, genetic instability, tissue invasion and metastasis (Harris, Nat. Rev. Cancer 2: 38-47, 2002; Maxwell et al., Curr. Opin. Genet. Dev. 11: 293-299, 2001).
HIF is a heterodimer composed of an alpha subunit complexed with a beta subunit, both of which are basic helix-loop-helix transcription factors. The beta subunit of HIF is a constitutive nuclear protein. The alpha subunit is the regulatory subunit specific to the oxygen response pathway, and can be one of three subunits, HIF1alpha, 2 alpha or 3 alpha (HIF1 , HIF2α and HIF3α, respectively) (Maxwell et al., Curr. Opin. Genet. Dev. 11: 293-299, 2001; Safran and Kaelin, J. Clin. Invest. 111: 779-783, 2003).
Until a tumor establishes a blood supply, hypoxic conditions limit tumor growth. Subsequent increases in HIF1α activity result in increased expression of target genes such as vascular endothelial growth factor (VEGF). VEGF expression is essential for vascularization and the establishment of angiogenesis in most solid tumors (Iyer et al., Genes Dev. 12: 149-162, 1998). A significant association between HIF1α, VEGF overexpression and tumor grade is also seen in human glioblastoma multiforme, the highest grade glioma, in which mean patient survival time is less than one year. The rapidly proliferating tumor outgrows its blood supply, resulting in extensive necrosis, and these regions express high levels of HIF1α protein and VEGF mRNA, suggesting a response of the tumor to hypoxia (Zagzag et al., Cancer 88: 2606-2618, 2000).
The gene encoding HIF2α (also, endothelial PAS domain protein 1, EPAS1, MOP2, hypoxia-inducible factor 2, HIF-related factor, HRF, HIF1 alpha-like factor, HLF) was initially identified as a transcription factor expressed in endothelial cells (Ema et al., Proc. Natl. Acad. Sci. U.S.A. 94: 4273-4278, 1997; Flamme et al., Mech. Dev. 63: 51-60, 1997; Hogenesch et al., J. Biol. Chem. 272: 8581-8593, 1997; Tian et al., Genes Dev. 11: 72-82, 1997). A link between elevated EPAS1 activity and angiogenesis has been demonstrated by experiments that show how HIF activity regulates VEGF expression. Normal human kidney cells typically have low levels of EPAS1, but upon introduction of a vector encoding EPAS1 into these cells, VEGF mRNA and protein levels increase significantly (Xia et al., Cancer 91: 1429-1436, 2001). When EPAS1 was inhibited, VEGF expression was significantly decreased, thus demonstrating a direct link between EPAS1 activity and VEGF expression (Xia et al., Cancer 91: 1429-1436, 2001). A correlation between HIF activity and VEGF expression is also observed in malignant cells and tissues. EPAS1 can be readily detected in renal cell carcinoma (RCC) cell lines in the absence of a vector encoding EPAS1 (Xia et al., Cancer 91: 1429-1436, 2001). Significant increases in EPAS1 and VEGF mRNA in renal cell carcinoma tissue samples, compared to normal tissue, suggest that abnormal activation of EPAS1 may be involved in the angiogenesis of RCC (Xia et al., Cancer 91: 1429-1436, 2001).
In addition to RCC, the expression of EPAS1 in other malignancies has also been reported. EPAS1 is expressed at the levels of mRNA and protein in human bladder cancers, especially in those with an invasive phenotype (Xia et al., Urology 59: 774-778, 2002). Another example of overexpression of EPAS1 is seen in squamous cell head-and-neck cancer (SCHNC). Higher levels of EPAS1 were associated with locally aggressive behavior of SCHNC, as well as intensification of angiogenesis (Koukourakis et al., Int. J. Radiat. Oncol. Biol. Phys. 53: 1192-1202, 2002). These findings also demonstrated a link between overexpression of EPAS1 and resistance to chemotherapy. Yet another correlation between overexpression of EPAS1 and cancer is seen in malignant pheochromocytomas, which exhibit a higher level of EPAS1 and an induced VEGF pathway, when compared to benign counterparts (Favier et al., Am. J. Pathol. 161: 1235-1246, 2002). EPAS1 overexpression is also a common event in non-small-cell lung cancer (NSCLC) and is related to the up-regulation of multiple angiogenic factors and overexpression of angiogenic receptors by cancer cells. EPAS1 overexpression in NSCLC is an indicator of poor prognosis (Giatromanolaki et al., Br. J. Cancer 85: 881-890, 2001). Elevated levels of EPAS1 mRNA and protein are seen in human lung adenocarcinoma cells, and exposure of these cells to hypoxia further increases EPAS1 expression (Sato et al., Am. J. Respir. Cell Mol. Biol. 26: 127-134, 2002). Taken together, these studies demonstrate that elevated EPAS1 confers aggressive tumor behavior, and that targeting the HIF pathway may aid the treatment of several different types of cancers.
Furthermore, the hypoxia response element plays a role in constitutively upregulating an isoform of VEGF in cancer cell lines under nonnoxic conditions. The HRE located within a cell type-specific enhancer element in glioblastoma cells participates in the up-regulation of VEGF expression through enhanced binding of EPAS1 to the HRE (Liang et al., J. Biol. Chem. 277: 20087-20094, 2002). A truncated version of EPAS1 that can bind to hypoxia-inducible factor 1 beta, but not to the HRE, was unable to transactivate the VEGF promoter (Liang et al., J. Biol. Chem. 277: 20087-20094, 2002). This further demonstrates the capability of cancer cells to combat hypoxic conditions by enhancing expression of factors required for vascularization and angiogenesis.
As a consequence of EPAS1 involvement in many diseases, there remains a long felt need for additional agents capable of effectively regulating EPAS1 function. Such inhibition is especially important in the treatment of cancer, given that the upregulation of expression of EPAS1 is associated with many different types of cancer.